1. Field of the Invention
The present invention relates to an immunoassay process of a target antibody or antigen in a specimen, which comprises mixing the specimen with an antigen or antibody corresponding to the target antibody or antigen to elicit immuno-agglutination reaction, and then detecting or determining the agglutination state. The present invention also relates to a reagent used in the assay and a method for producing the reagent.
2. Related Background Art
In clinical laboratory tests, various components in biological samples such as blood or urine are assayed to diagnose various diseases, for which various assay methods have been developed and used. For example, enzymatic assays using enzyme reaction and immunoassays using antigen-antibody reaction have widely been used. In recent years, especially, immunoassay utilizing highly specific antigen-antibody reactions has been used more frequently because of its high accuracy.
In the field of environmental and food analysis, usually the target substances are extracted from the sample, concentrated and then analyzed by high performance liquid chromatography or gas chromatography. However, in recent years, as seen with problems of dioxin or endocrine disrupting chemicals (so-called environmental hormones), assay of substances of extremely low concentrations, assay of a wide variety of target species, rapid assay of a large number of specimens, etc. are required more and more, which conventional methods have difficulty to cope with. Thus, also in the field of environmental and food analysis, immunoassay using antigen-antibody reaction is beginning to be used.
Examples of immunoassay include immunoagglutination assay (immunonephelometry and latex agglutination assay), enzyme immunoassay, and radioimmunoassay, which may be selected to suit the purposes. Immunoagglutination assay, especially, are widely used as a simple on-site assay method, because it not necessarily requires large expensive assay apparatuses, and is capable of an easy determination by visual observation. In immunoagglutination assay, an antigen or an antibody is immobilized on a water-insoluble carrier, and the carrier is mixed with a specimen to elicit immune agglutination reaction, and the level of agglutination is determined. Generally, polystyrene latex or gelatin particles are used as the water-insoluble carrier particles (Japanese Patent Application Laid-Open Nos. 11-295313 and 2000-338108). Since these materials are white or transparent, so that special techniques and trainings are required to carry out visual determination.
In order to solve the above problem, use of a colored insoluble carrier such as a pigment or dye has been proposed for easy visual determination. Japanese Patent Application Laid-Open No. 4-274762 discloses an immunoagglutination assay that uses phthalocyanine pigment as an insoluble carrier to which antibody is immobilized by physical adsorption. As a result, the agglutinate formed by the immune agglutination reaction has a blue color, enabling easy visual determination of the reaction.
However, when an antigen or antibody is immobilized on an insoluble carrier such as pigment by physical adsorption, sufficient adsorption may not be attained at times, causing problems regarding the production efficiency and storage stability of the assay reagent. Moreover, when an antigen or antibody is immobilized by physical adsorption, due to the difference of hydrophobicity of antigen or antibody, constant and quantitative immobilization of antigen or antibody is often difficult. This seriously affects performance of the assay system, and causes difference between production lots.
To solve this problem, a method has been proposed in which an antibody is partially denatured to increase its hydrophobicity before the antibody is adsorbed onto an insoluble carrier (Seikagaku Jikken Ho 11 (Biochemical Experiment Method 11), Enzyme Immunoassay, Tokyo Kagaku Dojin Co., Ltd., p. 270, 1989). However, this method has a problem that the antibody may lose its activity by denaturation so that this method can be applied not to all types of antibody.